Sulfonamide antibiotics prevent the biosynthesis of reduced folate compounds by inhibiting the production of H2pteroate from 6-hydroxymethylpterin pyrophosphate and p- aminobenzaoate (PABA). A common mode of resistance to sulfonamide antibiotics in E. coli is via mutation of the structural gene encoding H2pteroate synthase, so that the altered enzyme product is a sulfonamide resistant form that discriminates more effectively between PABA and sulfonamides than does the wild type enzyme. Sulfonamide resistance conferred by many R-determinant plasmids isolated from multiple antibiotic resistant clinical E. coli strains is by the same mechanism: expression of a sulfonamide resistant H2pteroate synthase. However, I have shown that sulfonamide resistance in E. coli can be mediated by gene amplification. Although I have not yet determined the identity of the amplified gene, the gene encoding H2pteroate synthase is a prime candidate, since that gene product is the target of sulfonamide inhibition. The sulfonamide resistant strain that I am characterizing has an 8-fold tandemly amplified DNA segment 18 kb in length. The association of an insertion sequence (IS5) with one (or both) endpoints of the amplified DNA suggests a role for site specific (illegitimate) recombination. This event is distinct from other illegitimate amplifications that seem to be independent of IS sequences. This application proposes to determine the mechanism of the gene amplification event, and to characterize H2pteroate synthase structure, function and expression at the genetic level.